N-Glycosylation influences transport, but not cellular trafficking, of a neuronal amino acid transporter SNAT1.

SNAT1 is a system N/A neutral amino acid transporter that primarily expresses in neurons and mediates the transport of l-glutamine (Gln). Gln is an important amino acid involved in multiple cellular functions and also is a precursor for neurotransmitters, glutamate and GABA. In the present study, we demonstrated that SNAT1 is an N-glycoprotein expressed in neurons. We identified three glycosylation sites at asparagine residues 251, 257 and 310 in SNAT1 protein, and that the first two are the primary sites. The biotinylation and confocal immunofluorescence analysis showed that the glycosylation-impaired mutants and deglycosylated SNAT1 were equally capable of expressing on the cell surface. However, l-Gln and 3H-labeled methyl amino isobutyrate (MeAIB) was significantly compromised in N-glycosylation-impaired mutants and deglycosylated SNAT1 when compared with the wild-type control. Taken together, these results suggest that SNAT1 is an N-glycosylated protein with three de novo glycosylation sites and N-glycosylation of SNAT1 may play an important role in the transport of substrates across the cell membrane.

Identification of a Disulfide Bridge in Sodium-Coupled Neutral Amino Acid Transporter 2(SNAT2) by Chemical Modification.

Sodium-coupled neutral amino acid transporter 2 (SNAT2) belongs to solute carrier 38 (SLC38) family of transporters, which is ubiquitously expressed in mammalian tissues and mediates transport of small, neutral amino acids, exemplified by alanine(Ala, A). Yet structural data on SNAT2, including the relevance of intrinsic cysteine residues on structure and function, is scarce, in spite of its essential roles in many tissues. To better define the potential of intrinsic cysteines to form disulfide bonds in SNAT2, mutagenesis experiments and thiol-specific chemical modifications by N-ethylmaleimide (NEM) and methoxy-polyethylene glycol maleimide (mPEG-Mal, MW 5000) were performed, with or without the reducing regent dithiothreitol (DTT) treatment. Seven single mutant transporters with various cysteine (Cys, C) to alanine (Ala, A) substitutions, and a C245,279A double mutant were introduced to SNAT2 with a hemagglutinin (HA) tag at the C-terminus. The results showed that the cells expressing C245A or C279A were labeled by one equivalent of mPEG-Mal in the presence of DTT, while wild-type or all the other single Cys to Ala mutants were modified by two equivalents of mPEG-Mal. Furthermore, the molecular weight of C245,279A was not changed in the presence or absence of DTT treatment. The results suggest a disulfide bond between Cys245 and Cys279 in SNAT2 which has no effect on cell surface trafficking, as well as transporter function. The proposed disulfide bond may be important to delineate proximity in the extracellular domain of SNAT2 and related proteins.

The SLC38 family of sodium-amino acid co-transporters.

Transporters of the SLC38 family are found in all cell types of the body. They mediate Na(+)-dependent net uptake and efflux of small neutral amino acids. As a result they are particularly expressed in cells that grow actively, or in cells that carry out significant amino acid metabolism, such as liver, kidney and brain. SLC38 transporters occur in membranes that face intercellular space or blood vessels, but do not occur in the apical membrane of absorptive epithelia. In the placenta, they play a significant role in the transfer of amino acids to the foetus. Members of the SLC38 family are highly regulated in response to amino acid depletion, hypertonicity and hormonal stimuli. SLC38 transporters play an important role in amino acid signalling and have been proposed to act as transceptors independent of their transport function. The structure of SLC38 transporters is characterised by the 5 + 5 inverted repeat fold, which is observed in a wide variety of transport proteins.

Identification of a disulfide bridge important for transport function of SNAT4 neutral amino acid transporter.

SNAT4 is a member of system N/A amino acid transport family that primarily expresses in liver and muscles and mediates the transport of L-alanine. However, little is known about the structure and function of the SNAT family of transporters. In this study, we showed a dose-dependent inhibition in transporter activity of SNAT4 with the treatment of reducing agents, dithiothreitol (DTT) and Tris(2-carboxyethyl)phosphine (TCEP), indicating the possible involvement of disulfide bridge(s). Mutation of residue Cys-232, and the two highly conserved residues Cys-249 and Cys-321, compromised the transport function of SNAT4. However, this reduction was not caused by the decrease of SNAT4 on the cell surface since the cysteine-null mutant generated by replacing all five cysteines with alanine was equally capable of being expressed on the cell surface as wild-type SNAT4. Interestingly, by retaining two cysteine residues, 249 and 321, a significant level of L-alanine uptake was restored, indicating the possible formation of disulfide bond between these two conserved residues. Biotinylation crosslinking of free thiol groups with MTSEA-biotin provided direct evidence for the existence of a disulfide bridge between Cys-249 and Cys-321. Moreover, in the presence of DTT or TCEP, transport activity of the mutant retaining Cys-249 and Cys-321 was reduced in a dose-dependent manner and this reduction is gradually recovered with increased concentration of H2O2. Disruption of the disulfide bridge also decreased the transport of L-arginine, but to a lesser degree than that of L-alanine. Together, these results suggest that cysteine residues 249 and 321 form a disulfide bridge, which plays an important role in substrate transport but has no effect on trafficking of SNAT4 to the cell surface.

Membrane topological structure of neutral system N/A amino acid transporter 4 (SNAT4) protein.

Members of system N/A amino acid transporter (SNAT) family mediate transport of neutral amino acids, including l-alanine, l-glutamine, and l-histidine, across the plasma membrane and are involved in a variety of cellular functions. By using chemical labeling, glycosylation, immunofluorescence combined with molecular modeling approaches, we resolved the membrane topological structure of SNAT4, a transporter expressed predominantly in liver. To analyze the orientation using the chemical labeling and biotinylation approach, the "Cys-null" mutant of SNAT4 was first generated by mutating all five endogenous cysteine residues. Based on predicted topological structures, a single cysteine residue was introduced individually into all possible nontransmembrane domains of the Cys-null mutant. The cells expressing these mutants were labeled with N-biotinylaminoethyl methanethiosulfonate, a membrane-impermeable cysteine-directed reagent. We mapped the orientations of N- and C-terminal domains. There are three extracellular loop domains, and among them, the second loop domain is the largest that spans from amino acid residue ∼242 to ∼335. The orientation of this domain was further confirmed by the identification of two N-glycosylated residues, Asn-260 and Asn-264. Together, we showed that SNAT4 contains 10 transmembrane domains with extracellular N and C termini and a large N-glycosylated, extracellular loop domain. This is the first report concerning membrane topological structure of mammalian SNAT transporters, which will provide important implications for our understanding of structure-function of the members in this amino acid transporter family.

A conserved Na(+) binding site of the sodium-coupled neutral amino acid transporter 2 (SNAT2).

The SLC38 family of solute transporters mediates the coupled transport of amino acids and Na(+) into or out of cells. The structural basis for this coupled transport process is not known. Here, a profile-based sequence analysis approach was used, predicting a distant relationship with the SLC5/6 transporter families. Homology models using the LeuT(Aa) and Mhp1 transporters of known structure as templates were established, predicting the location of a conserved Na(+) binding site in the center of membrane helices 1 and 8. This homology model was tested experimentally in the SLC38 member SNAT2 by analyzing the effect of a mutation to Thr-384, which is predicted to be part of this Na(+) binding site. The results show that the T384A mutation not only inhibits the anion leak current, which requires Na(+) binding to SNAT2, but also dramatically lowers the Na(+) affinity of the transporter. This result is consistent with a previous analysis of the N82A mutant transporter, which has a similar effect on anion leak current and Na(+) binding and which is also expected to form part of the Na(+) binding site. In contrast, random mutations to other sites in the transporter had little or no effect on Na(+) affinity. Our results are consistent with a cation binding site formed by transmembrane helices 1 and 8 that is conserved among the SLC38 transporters as well as among many other bacterial and plant transporter families of unknown structure, which are homologous to SLC38.

Regulation of amino acid transporter ATA2 by ubiquitin ligase Nedd4-2.

We report here that ubiquitin ligase Nedd4-2 regulates amino acid transporter ATA2 activity on the cell surface. We first found that a proteasome inhibitor MG132 increased the uptake of alpha-(methylamino)isobutyric acid, a model substrate for amino acid transport system A, in 3T3-L1 adipocytes as well as the preadipocytes. Transient expression of Nedd4-2 in Xenopus oocytes and Chinese hamster ovary cells down-regulated the ATA2 transport activity induced by injected cRNA and transfected cDNA, respectively. Neither the Nedd4-2 mutant with defective catalytic domain nor c-Cbl affected the ATA2 activity significantly. RNA-mediated interference of Nedd4-2 increased the ATA2 activity in the cells, and this was associated with decreased polyubiquitination of ATA2 on the cell surface membrane. Immunofluorescent analysis of Nedd4-2 in the adipocytes stably transfected with the enhanced green fluorescent protein (EGFP)-tagged ATA2 showed the co-localization of Nedd4-2 and EGFP-ATA2 in the plasma membrane but not in the perinuclear ATA2 storage site, supporting the idea that the primary site for the ubiquitination of ATA2 is the plasma membrane. These data suggest that ATA2 on the plasma membrane is subject to polyubiquitination by Nedd4-2 with consequent endocytotic sequestration and proteasomal degradation and that this process is an important determinant of the density of ATA2 functioning on the cell surface.

Sodium-coupled neutral amino acid (System N/A) transporters of the SLC38 gene family.

The sodium-coupled neutral amino acid transporters (SNAT) of the SLC38 gene family resemble the classically-described System A and System N transport activities in terms of their functional properties and patterns of regulation. Transport of small, aliphatic amino acids by System A subtypes (SNAT1, SNAT2, and SNAT4) is rheogenic and pH sensitive. The System N subtypes SNAT3 and SNAT5 also countertransport H(+), which may be key to their operation in reverse, and have narrower substrate profiles than do the System A subtypes. Glutamine emerges as a favored substrate throughout the family, except for SNAT4. The SLC38 transporters undoubtedly play many physiological roles including the transfer of glutamine from astrocyte to neuron in the CNS, ammonia detoxification and gluconeogenesis in the liver, and the renal response to acidosis. Probing their regulation has revealed additional roles, and recent work has considered SLC38 transporters as therapeutic targets in neoplasia.

Transcriptional control of the human sodium-coupled neutral amino acid transporter system A gene by amino acid availability is mediated by an intronic element.

System A amino acid transporter (SNAT2) gene expression is up-regulated at the transcriptional level in response to amino acid deprivation. Functional analysis of genomic fragments 5' upstream of the transcription start site, for both human and mouse SNAT2 genes showed that these regions exhibit promoter activity, but were amino acid unresponsive. However, when the human and mouse constructs were extended to include intron 1, it was observed that the rate of transcription was increased following amino acid deprivation. Deletion analysis of the human gene identified an intron 1 sequence spanning 54 nucleotides that was sufficient for conferring amino acid-dependent regulation to a minimal SNAT2 promoter. Alignment of the corresponding region from the human, mouse, and rat genomes revealed three highly conserved sequences. From site-directed mutagenesis, it was concluded that one of these sites functions as an amino acid response element (AARE) to regulate transcription. The core sequence of this site is identical to the AARE in the human CHOP gene. The SNAT2 AARE, along with a nearby conserved CAAT box, has enhancer activity in that it functions in an orientation and position independent manner, and it confers regulated transcription to a heterologous promoter.

Localization and functional relevance of system a neutral amino acid transporters in cultured hippocampal neurons.

Glutamine and alanine are important precursors for the synthesis of glutamate. Provided to neurons by neighboring astrocytes, these amino acids are internalized by classical system A amino acid carriers. In particular, System A transporter (SAT1) is a highly efficient glutamine transporter, whereas SAT2 exhibits broad specificity for neutral amino acids with a preference for alanine. We investigated the localization and the functional relevance of SAT1 and SAT2 in primary cultures of hippocampal neurons. Both carriers have been expressed since early developmental stages and are uniformly distributed throughout all neuronal processes. However, whereas SAT1 is present in axonal growth cones and can be detected at later developmental stages at the sites of synaptic contacts, SAT2 does not appear to be significantly expressed in these compartments. The non-metabolizable amino acid analogue alpha-(methylamino)-isobutyric acid, a competitive inhibitor of system A carriers, significantly reduced miniature excitatory postsynaptic current amplitude in neurons growing on top of astrocytes, being ineffective in pure neuronal cultures. alpha-(Methylamino)-isobutyric acid did not alter neuronal responsitivity to glutamate, thus excluding a postsynaptic effect. These data indicate that system A carriers are expressed with a different subcellular distribution in hippocampal neurons and play a crucial role in controlling the astrocyte-mediated supply of glutamatergic neurons with neurotransmitter precursors.